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Specific knockdown of OCT4 in human embryonic stem cells by inducible short hairpin RNA interference.

ABSTRACT: Manipulation of gene function in embryonic stem cells by either over expression or downregulation is critical for understanding their subsequent cell fate. We have developed a tetracycline-inducible short hairpin RNA interference (shRNAi) for human embryonic stem cells (hESCs) and demonstrated doxycycline dose-dependent knockdown of the transcription factor OCT4 and the cell surface antigen beta2-microglobulin. The induced knockdown of OCT4 resulted in rapid differentiation of hESCs with a significant increase in transcription of genes associated with trophoblast and endoderm lineages, the extent of which was controlled by the degree of induction. Transgene toxicity, which may occur in conditional over-expression strategies with hESCs, was not observed with wild-type Tet repressor protein. The system allows efficient, reversible, and long-term downregulation of target genes in hESCs and enables the generation of stable transfectants for the knockdown of genes essential for cell survival and self-renewal, not necessarily possible by nonconditional shRNAi methods. STEM CELLS 2009;27:776-782.

SUBMITTER: Zafarana G

PROVIDER: S-EPMC2847189 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Specific knockdown of OCT4 in human embryonic stem cells by inducible short hairpin RNA interference.

Zafarana Gaetano G Avery Stuart R SR Avery Katie K Moore Harry D HD Andrews Peter W PW

Stem cells (Dayton, Ohio) 20090401 4

Manipulation of gene function in embryonic stem cells by either over expression or downregulation is critical for understanding their subsequent cell fate. We have developed a tetracycline-inducible short hairpin RNA interference (shRNAi) for human embryonic stem cells (hESCs) and demonstrated doxycycline dose-dependent knockdown of the transcription factor OCT4 and the cell surface antigen beta2-microglobulin. The induced knockdown of OCT4 resulted in rapid differentiation of hESCs with a signi ...[more]

PMID: 19350677

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Project description:During hypoxia, upregulation of hypoxia inducible factor-1 alpha transcriptional factor can activate several downstream angiogenic genes. However, hypoxia inducible factor-1 alpha is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Here we hypothesize that short hairpin RNA (shRNA) interference therapy targeting PHD2 can be used for treatment of myocardial ischemia and this process can be followed noninvasively by molecular imaging.PHD2 was cloned from mouse embryonic stem cells by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted into the pSuper vector driven by the H1 promoter followed by a separate hypoxia response element-incorporated promoter driving a firefly luciferase reporter gene. This construct was used to transfect mouse C2C12 myoblast cell line for in vitro confirmation. Compared with the control short hairpin scramble (shScramble) as control, inhibition of PHD2 increased levels of hypoxia inducible factor-1 alpha protein and several downstream angiogenic genes by >30% (P<0.01). Afterward, shRNA targeting PHD2 (shPHD2) plasmid was injected intramyocardially following ligation of left anterior descending artery in mice. Animals were randomized into shPHD2 experimental group (n=25) versus shScramble control group (n=20). Bioluminescence imaging detected plasmid-mediated transgene expression for 4 to 5 weeks. Echocardiography showed the shPHD2 group had improved fractional shortening compared with the shScramble group at Week 4 (33.7%+/-1.9% versus 28.4%+/-2.8%; P<0.05). Postmortem analysis showed increased presence of small capillaries and venules in the infarcted zones by CD31 staining. Finally, Western blot analysis of explanted hearts also confirmed that animals treated with shPHD2 had significantly higher levels of hypoxia inducible factor-1 alpha protein.This is the first study to image the biological role of shRNA therapy for improving cardiac function. Inhibition of PHD2 by shRNA led to significant improvement in angiogenesis and contractility by in vitro and in vivo experiments. With further validation, the combination of shRNA therapy and molecular imaging can be used to track novel cardiovascular gene therapy applications in the future.

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Specific knockdown of OCT4 in human embryonic stem cells by inducible short hairpin RNA interference.

Publications

Specific knockdown of OCT4 in human embryonic stem cells by inducible short hairpin RNA interference.

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